Intracellular pathogens exploit a variety of host factors enabling them to proliferate and cause an array of infections. Listeria monocytogenes is an intracellular pathogen that is able to escape the bacteriocidal environment of the host cell vacuole into the cytosol where it can replicate and then spread to neighboring cells. L. monocytogenes'ability to escape from host vacuoles is facilitated by the pore forming toxin listeriolysin O (LLO). Although LLO is known to be activated in response to the acidic environment of pathogen-containing vacuoles (PCV), the model of pH activation does not completely explain LLO- dependent escape. Preliminary studies from our laboratory demonstrate that LLO-mediated vacuole escape is also regulated by the cystic fibrosis transmembrane conductance regulator (CFTR), a well-characterized chloride transporter. We hypothesize that chloride flux facilitates L. monocytogenes'ability to escape from PCV by modulating LLO activity. Chloride flux may mediate vacuole escape by one of two ways: vacuole acidification or by transiently altering the vacuolar salt concentration which, in turn, regulates LLO. We propose the following specific aims to understand the mechanism by which chloride flux facilitates LLO- mediated vacuole escape: 1) Use genetics, microscopy, and a mouse model of infection to define the role of CFTR-mediated chloride flux on the vacuole environment and escape of L. monocytogenes, 2) Measure gene expression, hemolytic activity, and oligomerization to determine how LLO is regulated in response to chloride flux, and 3) Employ confocal microscopy to identify the role of CFTR trafficking in intracellular infection. Our work has expanded significance in that CFTR-mediated chloride flux may provide essential factors to vacuolar pathogens which facilitate their ability to survive in these otherwise nutrient-limited compartments. PUBLIC HEALTH RELEVANCE: Infections caused by Listeria monocytogenes are most commonly foodborne and occur in immunocompromised individuals, including pregnant women. The purpose of this proposal is to characterize human host and bacterial factors which enable disease progression. Results obtained from this research may enable the development of therapeutics to treat these infections